Title

Author

Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

Qian Wang

Abstract

Plant viruses and virus-like protein cages (VLPs) have shown significant promise for a wide range of biomedical applications. In contrast to the artificial nanomaterials, VLPs exhibit unique properties including their well-organized three dimensional (3D) structures, monodispersity, high-polyvalent surface, and easily functionalized by genetic and chemical methods, which are difficult to be achieved by man-made materials. Therefore, the fabrication and the biomedical applications of biomaterials using viruses as building blocks will be studies in this work.

In this dissertation, I have done chemical modification of virus coat proteins to functionalize VLPs including rod-shaped virus (TMV, TVCV) and filamentous virus (PVX, TEV), and genetically engineered TMV coat protein by inserting an Arg-Gly-Asp peptide sequence (RGD) in order to display functional groups on the surface of TMV coat protein. Using these functionalized VLPs as building blocks, several methods have been studied to fabricate extracellular matrix (ECM) mimic substrates, which are well known to support for cellular adhesion, growth, and differentiation. The fibrous network of ECM structures can be mimetic by an electrospinning method. Using this technique, the functionalized plant viruses or ECM proteins have been electrospun with polymers, forming one dimentional (1D) nanofibers. The random distribution of nanofibers on a glass substrate resembles the ECM fibrous network with high porosity and surface-to-volume ratio. With various amount of TMV, the electrospun TMV and polyvinyl alcohol (PVA) nanofibers remain homogeneous without phase separation. The same result is obtained in the case of the as-spun polycaprolactone (PCL)-collagen. In biomedical applications, these electrospun ECM mimic substrates have been tested in vitro cell studies of baby hamster kidney (BHK) cells. As a result, in contrast to cells grown on PVA or PVA-TMV substrate, BHK cells show better adhesion and spreading on the RGD mutant TMV-PVA fibrous mats. Similarly, the breast cancer stem cell M605 also displayed better spreading on PCL-collagen substrates than that on the PCL fibrous matrix. In addition, on aligned electrospun fibrous mats, BHK cells were found to follow the alignment of the as-spun nanofiber.

Virus can also be self-assembled into ECM mimic substrates using a convective method. Driven by the interactions between the triple interfaces of air, solution, and glass surface, the filamentous virus -- bacteriophage M13 was self-assembled into a patterned M13 film. The topographic feature provides physical cues to promote BHK cells spreading and alignment in the direction of virus assembly. To characterize the cell-substrate interactions, cells were decellularized to expose the denuded ECM structure. Using Scanning Electron Microscopy (SEM) and fluorescence microscopy, the ECM proteins fibronectin and collagen (type Ι) were found to align on the M13 film while to randomize on a glass substrate.

In addition, functionalized VLPs have also shown capability to assemble with polymer poly 4-vinylpyridine (P4VP) into three dimensional (3D) core-shell nanostructures serving as a vehicle for drug delivery. To exploit the assembly of VLPs and polymers, a TEM thin-sectioning was studied to characterize VLP-P4VP 3D nanocomposites. By imaing thin sections of ferritin-P4VP and elemental characterization, we found that the 3D nanoparticle is composed of ferritins on the surface and P4VP staying inside to form a core. This polymer-VLP core-shell opens potential applications in drug delivery targeting tumor cells. Overall, functionalized viruses and VLPs have played important roles in regulating cell adhesion, spreading and tumor cells targeting.